Myeloid-derived suppressor cells (MDSCs) are a significant obstacle for immunotherapy of cancer. It is of great clinical relevance to study the mechanism of MDSCs accumulation in mouse spleens and establish a stable method to obtain high-purity MDSCs in vitro for further research. Here, we established a new method for amplifying a large number of highly pure MDSCs in vitro. To mimic the microenvironment of MDSCs development in vivo, mouse splenic stroma feeder cells and serum-free medium containing granulocyte-macrophage colony stimulating factor (GM-CSF) were used to induce myeloid precursors in mouse bone marrow cells, which differentiate into MDSCs. Development and immunological functions of the cells were monitored both in vivo and in vitro. A total of 4 × 108 MDSCs could be obtained from the bone marrow from one mouse, the ratio of CD11b+Gr-1+ MDSCs could reach 93.8% ± 3.3% after nine days of culture in vitro. Cultured MDSCs maintained a similar immunophenotype with MDSCs found in tumor-bearing mice. Colony forming assay in vitro and in vivo demonstrated that these were myeloid precursor cells. These cells generated high levels of reactive oxygen species and arginase 1 to prevent proliferation of CD8+ T cells in vitro. These also increased regulatory T (Treg) cells in blood while promoting the growth of lymphoma in vivo. In addition, cultured MDSCs effectively inhibited acute graft-versus-host disease (aGVHD). Our findings suggest that mouse splenic stroma plays an important role in the generation of MDSCs and represent a preliminary mechanism for the accumulation of MDSCs in spleens, and thereby lay the foundation for basic research and the clinical application of MDSCs.

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Aims: Recent studies have revealed that neutrophil extracellular traps (NETs) provide negative feedback in the progression to chronic inflammation and contribute to the pathogenesis of multiple autoimmune diseases including type 1 diabetes (T1D). In addition, accumulating evidences suggest that gut immunity play a key role in T1D pathogenesis. Our study aimed to evaluate whether staphylococcal nuclease (SNase) targeting intestinal NETs can ameliorate the intestinal inflammatory environment and protect against T1D development in non-obese diabetic(NOD) mice. Main methods: Degradation of NETs with SNase in vitro was examined using SYTOX green assay. NOD/LtJ mice were oral administration of Lactococcus lactisl (L. lactis) pCYT: SNase and blood glucose levels were monitored weekly. Several biomarkers of NETs formation, gut leakage and inflammation were determined using a commercial ELISA kit. T Cell phenotypes in peripheral immune system were analyzed in flow cytometry and fecal samples were isolated to investigate intestinal microbiota. Key findings: The oral delivery of SNase by L. lactis can decrease the NETs levels and ameliorate inflammation both in the intestine and pancreatic islets and finally effectively regulate the blood glucose levels of NOD mice. Meanwhile, zonulin and lipopolysaccharide levels also reduced in SNase-fed NOD mice, suggesting SNase could improve gut barrier function via intestinal NETs degradation. Furthermore, the abundances of the intestinal microbiota and butyrate-producing gut bacteria were also increased with SNase treatment. Significance: SNase shows potential for intestinal NETs to prevent T1D based on the gut-pancreas axis.

BACKGROUND:The aim of this study was to investigate the association of the formation of neutrophil extracellular traps (NETs) with gut leakage in type 1 (T1D) and type 2 diabetes (T2D). METHODS:In all, 105 subjects (56 T1D, 49 T2D) were included in the study. Eight biomarkers of NET formation and gut leakage (ie, protein arginine deiminase type 4 [PAD4], neutrophil elastase [NE], proteinase 3 [PR3], complement 5a [C5a], α1 -antitrypsin [AAT], DNase I, zonulin, and lipopolysaccharide [LPS]) were measured in serum samples by ELISA. Neutrophils were isolated and stimulated by phorbol myristate acetate to form NETs in vitro. Neutrophil intracellular contents were then collected and used as antigens to detect anti-neutrophil cytoplasmic antibodies (ANCA) in the serum. RESULTS:There was an increase in NET-associated proteins (PAD4, NE, PR3, C5a, AAT and DNase I) in new-onset T1D patients but not in those with T2D. Of PAD4, NE, and PR3, PAD4 was found to be the most sensitive biomarker for the diagnosis of T1D. Furthermore, circulating levels of zonulin and LPS were not only increased, but were also strongly correlated with NET formation and ANCA generation in T1D patients. CONCLUSIONS:This study provides evidence that increased formation of NETs, particularly PAD4, is closely associated with gut leakage in T1D but not T2D, and suggests that microorganisms and the release of neutrophil cytoplasmic antigen during the formation of NETs may be involved in the pathogenesis of T1D.

A major source of β cell generation is pancreatic progenitor-like cell differentiation. Multiple studies have confirmed that stem cell metabolism plays important roles in self-renewal and proliferation. In the absence of glucose, glutamine provides the energy for cell division and growth. Furthermore, α-ketoglutarate (αKG), a precursor for glutamine synthesis, is sufficient for enabling glutamine-independent cell proliferation. We have demonstrated that αKG contributes to the large-scale proliferation of pancreatic progenitor-like cells that can provide an ample amount of clinically relevant β cells. We compared the mRNA expression of a subset of genes, the abundance of ATP, reactive oxide species, mitochondrial number, and the colony-forming frequency between mouse pancreatic CD133+ and CD133− cells. We employed Real-Time PCR, immunostaining and passage assays to investigate self-renewal and proliferation of pancreatic progenitor-like cells in a 3D culture system in the presence and absence of αKG. The energy metabolism of CD133+ cells was more prone to oxidative phosphorylation. However, in the 3D culture system, when αKG was supplemented to the culture medium, the proliferation of the pancreatic progenitor-like cells was significantly elevated. We confirmed that the presence of αKG correlated with the up-regulation of Ten-Eleven Translocation (Tet). αKG can promote the proliferation of pancreatic progenitor-like cells via the up-regulation of Tet.

To further evaluate the effect of miR-217 in the proliferation of mouse adult pancreatic stem cells, we firstly transfected adult pancreatic stem cells with miR-217 mimics and studied the effect of miR-217 on proliferation through Western blot and immunofluorescence. Results showed that during the proliferation of adult pancreatic stem cells, miR-217 inhibited the protein expression of Ki-67 and Cyclin Dl, which are related to cell propagation. As well as that, to investigate the target genes of miR-217 and their conserved sites bound by the seed region of miR-217, we used bioinformatic algorithms to find a potential target of miR-217 and verified by dual-luciferase activity assay. Surprisingly, dual-luciferase activity assay revealed that miR-217 could decrease PMIR-REPORT-Sirt1-3'UTR luciferase activity and Sirt1 is a direct target of miR-217. Finally, we verified the function of Sirt1 in the proliferation of pancreatic stem cells. Overexpressionion of miR-217 in pancreatic stem cells inhibited the level of Sirt1 in protein level but not in mRNA level. Furthermore, activator of Sirt1 played positive effect on colony formation ability and cell proliferation and inhibitor of Sirt1 showed the opposite function. In conclusion, miR-217 inhibits the proliferation of mouse adult pancreatic stem cells through Sirt1 and decreased expression of miR-217 to contribute to the pancreatic stem cells development.

1-Methyl-D-tryptophan (1-MT) is extensively utilized in preclinical trials to deplete indoleamine 2,3-dioxigenase (IDO) activity and kynurenine pathway. Since IDO related signaling pathways aren’t well understood, some clinical reports affirmed IDO inhibiting therapeutic significance. Therefore, we did use direct tumor autologous antigens vaccination and 1-MT without chemotherapy to explore biological mechanisms and immunomodulations of 1-MT that motivate antitumor responses. However, DCs antigen-uptake capability, anti-tumor efficiency, intra-tumor and intracellular cytokines were assessed. Besides, CD133+ cells viability and tumor biomarkers were investigated. Splenocytes responses and their signaling pathways such TLRs 2 to 9, NF-κβ1-2, Wnt/β-catenin and TGF-β were dissected. Results evinced that a regimen of 1-MT and TAAs significantly reduced CSC CD133 + viability inside tumor microenvironment, besides increasing tumor cells necrosis and apoptosis. Expression of TGF-β, IDO, RANTES, and PDL-1 was also significantly reduced. Interestingly, 1-MT enhanced lymphocytes TLR2, TLR7, TLR8, and TLR9 pathways. It motivated lymphocytes’ NF-κβ2, STAT3, and STAT4 pathways, while reduced tumors’ NF-κβp65 and Wnt/β-catenin signaling pathways. We found that periphery and intra-tumor Treg cells were significantly decreased. In conclusion, depletion of indoleamine 2,3-dioxigenase activity evidenced IDO relation with tumor stem cells proliferation pathways. Furthermore, 1-MT supports immunotherapeutic vaccines susceptibility and tumor specific targeting by reducing tumorgensis signaling pathways.

Xanthine oxidase (XOD) is a key enzyme that catalyzes xanthine to uric acid. Most of the urate-lowering medicines targeting XOD have a limited effect on alleviating inflammation in spite of significant effects on decreasing serum uric acid level. In this study, we produced and characterized a novel monoclonal antibody (Anti-XOD mAb) using hybridoma technology based on a novel peptide OI5P-1(O-IA2(5)-P2-1),which containing a B-cell epitope of XOD and a novel Th2 built-in adjuvant I5P-1(IA2(5)-P2-1). Results of western blotting and cross-reactivity assay indicated that the mAb binds specifically to XOD and the affinity was 2.523×1010L/mol. The mAb reduced serum uric acid level and hepatic xanthine oxidase activity in potassium oxonate induced mice. A decreased methane dicarboxylic aldehyde level and an improved superoxide dismutase level in mAb treated mice indicated anti-lipid peroxidation effects of the mAb. Moreover, the mAb showed a significant immunomodulatory effect which could shift Th1/Th2 balance to Th2-dominant immunity. The mAb treatment alleviates inflammation induced by potassium oxonate, superior to the small molecule allopurinol treatment. For the first time, these results showed that the anti-XOD mAb may serve as a promising therapeutic approach for inflammatory response related to uric acid.

ABSTRACT Breast cancer stem cells (BCSCs), a small subset of breast cancer cells with stem cell–like properties, are essential in tumor formation, metastasis, resistance to anticancer therapies, and cancer recurrence. MicroRNAs (miRNAs) are involved in tumorigenicity by regulating specific oncogenes and tumor‐suppressor genes, and their roles in BCSCs are becoming apparent. A novel, 3‐dimensional (3D), semisolid culture system was established to culture MCF‐7 spheroid cells with high percentage of BCSCs. The differences in miRNA expression among the MCF‐7 parental cells, BCSC‐enriched MCF‐7 spheroid cells, and CD44 + /CD24 − MCF‐7 cells were evaluated by miRNA microarray, and the high expression of miR‐210 in MCF‐7 spheroid cells and CD44 + /CD24 − MCF‐7 cells was verified by quantitative RT‐PCR. MCF‐7 cells were cultured in a hypoxic chamber to detect the effect of hypoxia on miR‐210 expression and the sternness of the cells. The 3‐(4,5‐dimethylthiazol‐2‐ yl )‐2,5‐diphenyl tetrazolium bromide (MTT), transwell, and sphere‐formation assays were performed to detect the proliferation, migration, and self‐renewal ability of miR‐210– overexpressed MCF‐7 cells and MCF‐7 spheroid cells with miR‐210 knocked down. The target of miR‐210 was validated with a dual‐luciferase reporter assay and Western blotting. In vivo xenograft assay and metastasis assay were performed to study the effects of miR‐210 targeting E‐cadherin on BCSCs growth and lung metastasis, and the tumors were assessed by immunohistochemistry and immunofluorescence. We developed a novel 3D, semisolid culture system to culture MCF‐7 spheroid cells, which are enriched in BCSCs, and found, by performing miRNAs expression profiling, miR‐210 was up‐regulated in those cells compared with MCF‐7 parental cells. High miR‐210 expression was also detected in CD44 + /CD24 − MCF‐7 cells and human CD44 + /CD24 − breast cancer cells, which was demonstrated to be partially due to the hypoxic microenvironment around BCSCs in MCF‐7 spheroids or solid tumors. Ectopic expression of miR‐210 in MCF‐7 cells promoted their migration, invasion, proliferation, and selfrenewal in both in vitro and in vivo studies. We further reported that miR‐210 suppressed E‐cadherin expression by targeting the open reading frame region of E‐cadherin mRNA and by up‐regulation of E‐cadherin transcription repressor, Snail. Accordingly, E‐cadherin overexpression compromises the migration, invasion, proliferation, and self‐renewal ability of miR‐210–overexpressed MCF‐7 both in vitro and in vivo . These findings reveal a novel regulatory pathway centered on hypoxia‐mediated miR‐210 targeting of E‐cadherin, which contributes to the properties and breast tumorigenesis of BCSCs.—Tang, T., Yang, Z., Zhu, Q., Wu, Y., Sun, K., Alahdal, M., Zhang, Y., Xing, Y., Shen, Y., Xia, T., Xi, T., Pan, Y., Jin, L. Up‐regulation of miR‐210 induced by a hypoxic microenvironment promotes breast cancer stem cell metastasis, proliferation, and self‐renewal by targeting E‐cadherin. FASEB J. 32, 6965–6981 (2018). www.fasebj.org

In this study, the fungicidal effects and detailed action of chitosan against Ceratocystis fimbriata were evaluated. The results demonstrated that chitosan exhibited strong antifungal activity that restricted the mycelium extension and changed the hyphal morphology. Fluorescein diacetate (FDA) and propidium iodide (PI) double-staining directly visualized decreased cell viability in response to chitosan treatment. Investigation of the PI influx showed that chitosan induced irreversible cell membrane damage. The efflux of potassium ions from the cytosol into the extracellular matrix demonstrated that chitosan induced the leakage of intracellular components. Massive intracellular bis-(1, 3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] accumulation indicated the dissipation of membrane potential. Furthermore, chitosan clearly decreased the activity of H+/K+ ATPase. Fluorescence microscopy revealed that the fluorescence distribution and intensity of fluorescein isothiocyanate (FITC) changed as the incubation time increased. These results indicate that chitosan exerts a fungicidal effect via its ability to disturb fungal membranes.

Background/Aims: Pancreatic cancer remains one of the deadliest human malignancies, the lethality of which may be attributed to the presence of pancreatic cancer stem cells (PCSCs), a small subpopulation of cells existing within pancreatic tumor with high carcinogenesis. Therefore, it is crucial to establish an efficient enrichment and culture system of PCSCs and identify the key genes involved in the regulation of PCSCs. The three-dimensional (3D) liquid suspension mammosphere culture system has been established for enrichment and culture of PCSCs in vitro as the cell spheres are likely to originate from individual cell clone, but it has been challenged because the cell spheroids could be a result of cell aggregation. Methods: We optimized the existing culture system by adding methylcellulose to create a 3D semi-solid system which prevented the non-specific aggregation. Then we identified the CSC properties of Panc-1 spheroid cells cultured by this system by detecting the genes associated with stemness and by evaluation of the tumorigenicity in vitro and in vivo through invasion, migration and xenograft experiments methods. Subsequently, we performed high-throughput sequencing (HTS) of the Panc-1 spheroid cells. Results: We confirmed the PCSCs properties and high malignancy of the Panc-1 spheroid cells enriched by our novel 3D semi-solid system both in vitro and in vivo. Hundreds of mRNA, microRNA (miRNA) and dozens of long non-coding RNA (LncRNA) were identified to be differentially regulated in PCSCs-like Panc-1 spheroid cells compared with their parental cells by HTS. Conclusions: Our results demonstrate an efficient enrichment and culture system for Panc-1 spheroid cells with the PCSCs properties. The differentially expressed genes and their targets identified by the HTS of the Panc-1 spheroid cells can serve as new potential biomarkers for pancreatic cancer diagnosis and targeted therapy.

[基金项目] 江苏省社会发展-临床前沿技术资助项目(No. BE2016809);南京市科技发展计划项目资助(No. 201503011)。Project supported by the Foundation of the Jiangsu Social Development Clinical Frontier Technology (No. BE2016809), and the Foundation of the Nanjing Science and Technology Development Plan (No. 201503011) [作者简介] 李剑(1985-),男,博士生,主要从事肿瘤免疫治疗相关的基础研究,E-mail:lijian_cpu@126.com [通信作者] 朱学军(ZHU Xuejun,corresponding author),博士,主任医师,硕士生导师,主要从事肿瘤综合治疗相关研究,E-mail:zhuxj2@sina. com;刘根桃(LIU Gengtao,corresponding author),博士,教授,博士生导师,主要从事肿瘤干细胞与免疫治疗相关研究,E-mail:liugt@hotmail. com。△为共同通信作者 嵌合抗原受体修饰的CD19-CAR-T的体外构建、扩增及初步功能鉴定

Therapeutic efficacy of P277 against type 1 diabetes was extensively investigated and clinically evidenced. Clinical trials Phases I and II concluded promising results, while the data of P277 immunogenicity in Phase III trials represented weak responses that led to abolish medical use. But, a therapeutic performance of P277 cannot be forgotten. So, in order to exploit its therapeutic benefits and improve its immunogenicity, we developed a new analogue VP to optimize therapeutic efficacy and enhancing immunosuppressive modulations. However, new analogue was purified, and then used to immunize diabetic NOD mice to investigate antidiabetic effects through modulation of immunological status. So, DCs immune responses, relative TLRs, MyD88, and NF-K1 mRNA expression on DCs and splenocytes under VP effect were tested. Circulating and intracellular cytokines were also evaluated at treated and non-treated mice. Splenic T lymphocytes proliferation (Th1 and Treg cells) were also determined. Results revealed that VP significantly down regulates DCs maturation through TLR2, TLR4, and MyD88 pathways. It also shifts DCs to a tolerogenic polarization through NF-K1 pathway that mediates Th1 immunosuppression and enhances iTreg expanding in type1diabetes mice. Meanwhile, we noticed that VP significantly enhances iTreg CD25+FoxP3+ proliferation. In conclusion, VP showed promising immune potential to modulate immune regulatory responses and shifts DCs to suppress autoreactive Th1 cells which ameliorated immunosuppressive potency in the type1 diabetic mice.

P277 is a 24 amino-acids peptide, residues 437-460 of human heat shock protein 60 (HSP60). P277 or sequence repeated 6 x P277 was previously found showing potency preventive and therapeutic anti-diabetes functions in NOD mice, but aroused atherosclerosis due to the induction of anti-HSP65 autoantibodies as reported. To determine the intrinsic B epitope sequence, we screened P277 with pepscan method and then proved by detection of sera IgG from peptide fragments vaccinated mouse and rabbits. Results indicated HSP60 443-448 (ALLRCI) is potential intrinsic B epitope sequence of P277. We modified P277 by deleting the former three amino acids of ALLRCI (VP) or replacing these six with alanine (AP). The detection of serum lipid parameter in NOD mice and aorta endothelial damage levels in high-cholesterol diets fed rabbits demonstrated that VP induced higher antidiabetes efficacy and caused less arteriosclerosis-liked diseases separately. With less TLR2/4 activation of dendritic cells and macrophages, VP treatment reduced Th1 related P277 specific pro-inflammatory cytokines production and increased regulatory immune responses both in vivo and in vitro. These results indicated that optimized VP peptide might serve as a promising candidate for mouse type 1 diabetes therapy.

Background/Aims: Abnormal glucose metabolism and lipid metabolism are two key issues in Type 1 diabetes mellitus (T1DM). Insulin can control carbohydrate metabolism adequately, but cannot regulate lipid metabolism well in patients with T1DM. Glucagon-like peptide-1 receptor agonists (GLP-1RAs) have cured type 2 diabetes mellitus in clinical trials and have improved T1DM glycemic control in preclinical studies. However, previous studies have not reported whether GLP-1 can lower the serum concentration of non-esterified fatty acids (NEFAs). In this study, we examine whether GLP-1 can affect serum NEFA levels. Methods: The bioactivity of EGLP-1 (a novel GLP-1 analog) in vitro was analyzed in CG-HEK293 cells and with high-performance liquid chromatography. An intraperitoneal glucose tolerance test (IPGTT) was used to analyze the acute and sustained hypoglycemic effects of EGLP-1 in normal C57BL/6J mice. Streptozotocin-induced hyperglycemic mice were used to study the effects of EGLP-1 on blood glucose and NEFAs as well as its mechanism. Results: EGLP-1 activated GLP-1R and resisted dipeptidyl peptidase-IV digestion in vitro. Additionally, EGLP-1 had an insulinotropic action in vivo that lasted for approximately 6 h. In Streptozotocin-induced hyperglycemic mice, EGLP-1 improved hyperglycemia, inhibited food intake, and increased β-cell area. Serum physiological indexes showed that insulin and C-peptide levels were increased, while NEFA and triacylglycerol concentrations were decreased. Western blot analysis revealed that EGLP-1 significantly reduced phosphorylated-hormone sensitive lipase (pHSL) levels in white adipose tissue. Conclusions: EGLP-1 can improve hyperglycemia by increasing islet β-cell area and improving β-cell function, possibly due to reduced NEFA content in serum by lowering pHSL levels.

Magnetic nanoparticles (NPs) of superparamagnetic iron oxide (SPIO) have been explored for different kinds of applications in biomedicine, mechanics, and information. Here, we explored the synthetic SPIO NPs as an adjuvant on antigen cross-presentation ability by enhancing the intracellular delivery of antigens into antigen presenting cells (APCs). Particles with different chemical modifications and surface charges were used to study the mechanism of action of antigen delivery. Specifically, two types of magnetic NPs, γFe2O3/APTS (3-aminopropyltrimethoxysilane) NPs and γFe2O3/DMSA (meso-2, 3-Dimercaptosuccinic acid) NPs, with the same crystal structure, magnetic properties, and size distribution were prepared. Then, the promotion of T-cell activation via dendritic cells (DCs) was compared among different charged antigen coated NPs. Moreover, the activation of the autophagy, cytosolic delivery of the antigens, and antigen degradation mediated by the proteasome and lysosome were measured. Our results indicated that positive charged γFe2O3/APTS NPs, but not negative charged γFe2O3/DMSA NPs, enhanced the cross-presentation ability of DCs. Increased cross-presentation ability induced by γFe2O3/APTS NPs was associated with increased cytosolic antigen delivery. On the contrary, γFe2O3/DMSA NPs was associated with rapid autophagy. Overall, our results suggest that antigen delivered in cytoplasm induced by positive charged particles is beneficial for antigen cross-presentation and T-cell activation. NPs modified with different chemistries exhibit diverse biological properties and differ greatly in their adjuvant potentials. Thus, it should be carefully considered many different effects of NPs to design effective and safe adjuvants.

BACKGROUND:Transplantation of insulin-producing cells is considered an important diabetes therapy. Many research studies have shown that insulin-producing cells can be derived from the in-vitro cultured pancreatic colonies with self-renewal ability and multilineage potential. Even though these progenitor-like colonies have been prepared from adult pancreas cells, the efficient culture method is hardly established and regulation of the colonies is rarely known. We confirmed previously that single cells acquired from adult mouse pancreas could form cyst-like colonies in a 3D semi-solid system containing Matrigel and methylcellulose. These colonies could be passaged continuously without losing progenitor-like capacity. In the previous culturing system, however, conditioned medium from murine embryonic-stem-cell-derived pancreatic-like cells was used. This unregulated ingredient may reduce repeatability and affect following study. Thus, a new culturing system with certain components needs to be developed.METHODS:Single cell suspension was acquired from adult mouse pancreas and cultured in a Matrigel-based 3D system with epidermal growth factor, Nicotinamide, B27, and Noggin to form ring colonies. Serial-passage assay was performed to evaluate self-renewal ability. Real-time polymerase chain reaction and immunostaining were used to detect the expression of progenitor-related genes. A 2D differentiation method was used to testify the multilineage potency of the colonies. High-throughput sequencing (HTS) of the colonies was performed to profile the differentially expressed genes.RESULTS:We developed a 3D culturing system deprived of conditioned medium to propagate those colonies with high proliferative efficiency. HTS of the transcriptome of mRNAs, microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) showed differentially expressed genes compared to the whole pancreas (as control). In mRNAs, several surface marker genes were identified in the colonies. Moreover in noncoding RNAs, miR-21a, miR-31 and miR-155 were upregulated and miR-217, miR-802 and miR-375 were downregulated in colonies along with a number of other miRNAs and lncRNAs.CONCLUSIONS:Our results offer an efficient culture system for pancreatic progenitor-like colonies and HTS of the colonies serves as a target resource for following study of in-vitro cultured pancreatic progenitors. These findings should also contribute to our understanding of the transcriptional regulation of these progenitor-like colonies and the mechanisms behind their functions.

Hyperuricemia (HUA) is related to diabetes. Uric acid-induced inflammation and oxidative stress are risk factors for diabetes and its complications. Human urate transporter 1 (URAT1) regulates the renal tubular reabsorption of uric acid. IA-2(5)-P2-1, a potent immunogenic carrier designed by our laboratory, can induce high-titer specific antibodies when it carries a B cell epitope, such as B cell epitopes of DPP4 (Dipeptidyl peptidase-4), xanthine oxidase. In this report, we describe a novel multi-epitope vaccine composing a peptide of URAT1, an anti-diabetic B epitope of insulinoma antigen-2(IA-2) and a Th2 epitope (P2:IPALDSLTPANED) of P277 peptide in human heat shock protein 60 (HSP60). Immunization with the multi-epitope vaccine in streptozotocin-induced diabetes C57BL/6J mice successfully induced specific anti-URAT1 antibody, which inhibited URAT1 action and uric acid reabsorption, and increased pancreatic insulin level with a lower insulitis incidence. Vaccination with U-IA-2(5)-P2-1 (UIP-1) significantly reduced blood glucose and uric acid level, increased Th2 cytokines interleukin (IL)-10 and IL-4, and regulated immune reactions through a balanced Th1/Th2 ratio. These results demonstrate that the URAT1-based multi-epitope peptide vaccine may be a suitable therapeutic approach for diabetes and its complications.

Type 1 diabetes is a chronic organ-specific autoimmune disease in which selective destruction of insulin-producing β cells leads to impaired glucose metabolism and its attendant complications. IA2(5)P2-1, a potent immunogenic carrier which designed by our laboratory, can induce high titer specific antibodies when carry a B cell epitope, such as B cell epitopes of DPP4, xanthine oxidase, and Urate transporter protein. In this report, we describe a novel multi-epitope vaccine composing a peptide of DPP4, an anti-diabetic B epitope of Insulinoma antigen-2(IA-2) and a Th2 epitope (P2:IPALDSLTPANED) of P277 peptide in human heat shock protein 60 (HSP60). Immunization with the multi-epitope vaccine in non-obese diabetic (NOD) mice successfully induced specific anti-DPP4 antibody, inhibited plasma DPP4 activity, and increased serum GLP-1 level. Moreover, this antibody titer was correlated with the dose of immunization (20μg, 100μg). Inoculation of this vaccine in NOD mice significantly control blood glucose level, improved glucose excursion and increased insulin level in vivo. Consistent with a lower diabetic and insulitis incidence, a induced splenic T cells proliferation and tolerance were observed. IFN-γ secretion reduced and IL-10 increased significantly in the D41-IA2(5)-P2-1 treated mice compared to P277 and control group due to the potential immunomodulatory effect of the epitope in the vaccine. Immunohistochemical analysis and cytometry showed a rebalance of Th1/Th2 in NOD mice. Our results demonstrate that this multi-epitope vaccine may serve as a promising therapeutic approach for type 1 diabetes.

Tumor-derived autophagome (DRibble) is an effective therapeutic cancer vaccine inducing T cell recognition and death of tumor cells in mice. However, the potential for improved anti-tumor response still remains. Our previous study demonstrated that two repeats of a mycobacterial HSP70 407–426 (M2) peptide acted as adjuvant in improving anti-tumor efficacy of human umbilical vein endothelial cell (HUVEC) vaccine. Here, a DRibble vaccine conjugated with M2 (DRibble-M2) was designed as a novel vaccine to enhance anti-tumor activity. Compared with DRibble alone, DRibble-M2 vaccination more significantly inhibited the growth of mouse Lewis lung cancer both in a subcutaneous tumor model and in a lung metastasis model. Higher expression of antigen-specific CTL was induced by DRibble-M2. DRibble-M2 induced higher CD83 and CD86 expression in DC2.4 and also improved the internalization of DRibble antigen into DC2.4. Our data indicated that DRibble-M2 is a potential vaccine for clinical cancer therapy.